Additional file 3 of ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways

Additional file 3: Figure S3. HE (scale bar = 200 μm) and Masson (scale bar = 50 μm) staining in the rat calvarial defect model (n = 6)

Additional file 1 of ETV2 promotes osteogenic differentiation of human dental pulp stem cells through the ERK/MAPK and PI3K-Akt signaling pathways

Additional file 1: Figure S1. The mRNA level of CBFA1 in qRT-PCR (a) and RNA-Seq (b). *P < 0.05

DataSheet1_Chirality-biased protein expression profile during early stages of bone regeneration.docx

Introduction: Chirality is a crucial mechanical cue within the extracellular matrix during tissue repair and regeneration. Despite its key roles in cell behavior and regeneration efficacy, our understanding of chirality-biased protein profile in vivo remains unclear.Methods: In this study, we characterized the proteomic profile of proteins extracted from bone defect areas implanted with left-handed and right-handed scaffold matrices during the early healing stage. We identified differentially-expressed proteins between the two groups and detected heterogenic characteristic signatures on day 3 and day 7 time points.Results: Proteomic analysis showed that left-handed chirality could upregulate cell adhesion-related and GTPase-related proteins on day 3 and day 7. Besides, interaction analysis and in vitro verification results indicated that the left-handed chiral scaffold material activated Rho GTPase and Akt1, ultimately leading to M2 polarization of macrophages.Discussion: In summary, our study thus improved understanding of the regenerative processes facilitated by chiral materials by characterizing the protein atlas in the context of bone defect repair and exploring the underlying molecular mechanisms of chirality-mediated polarization differences in macrophages.</p

PTH promotes the repair of SAON after CD surgery in the bone tunnel and bone marrow of each group.

<p>(A) PTH enhanced bone formation, increased the number of osteoblasts (green arrows), reduced necrosis foci (red arrows), and decreased the number of mononuclear cells and empty lacunas (yellow arrows) in the distal femurs with steroid-induced osteonecrosis after CD. (B) in the coronal plane of distal femur with a bone tunnel (yellow dotted circles). (C) The percentage of the area of necrosis foci and the percentage of empty osteocyte lacuna were measured, n = 6 in the SAON-CD-PTH group, n = 6 in the SAON-CD group, and n = 4 in the sham-CD group, Data are presented as mean ± SD, and error bars in the figure denote SD, *p<0.05, **p<0.01 (H&E, 50x and 200x).</p

PTH improves the efficacy of CD in the treatment of SAON.

<p>(A) MR images of the distal femurs from each group (n = 4 in the Sham-CD group, n = 6 in the SAON-CD group and n = 6 in the SAON-CD-PTH group) showed normal signal intensity in the Sham-CD group. Focal inhomogeneous high SI in the T2-weight images (red arrows) and focal inhomogeneous low SI in the T1-weight in the SAON group. However, in the SAON-CD-PTH group, less high SI in the T2 weight image and less abnormal SI in the T1 weight image were revealed the ER in the T2 weight MR images (B) and the area of abnormal SI (C) were measured for the quantification of MR images.</p

PTH upregulates bone formation in SAON after CD surgery.

<p>Bone was labeled during regeneration and remodeling by calcein (green) and andalizarin red (red) (A). (B-C) The MS/BS and the MAR were measured and presented. Data are presented as mean ± SD, and error bars in the figure denote SD, *p<0.05, **p<0.01.</p

PTH improves revascularization both in the tunnel and in the bone marrow.

<p>Vessels (red arrows) from each group (n = 6 in the SAON-CD-PTH group, n = 6 in the SAON-CD group, and n = 4 in the sham-CD group) were visualized with α-SMA staining (200x) within the tunnel (A) and in the marrow (B). (C) The number per area of tissue and (D) the diameter of blood vessels were measured. Data are presented as mean ± SD, and error bars in the figure denote SD, *p<0.05, **p<0.01.</p

Study design and number of animals utilized at different stages.

<p>ON = osteonecrosis; LPS = lipopolysaccharide; MPS = methylprednisolone; SAON = steroid-associated osteonecrosis.</p

MRI images and histological analysis confirmed successful establishment of the SAON model.

<p>(A) MRI images of femurs on T1 and T2 weight from the SAON group (n = 4) and the Sham group before and 2 weeks after steroid administration, exhibited diffused and decreased SI (red arrows) in T1W. Focal hypointense lesion (green arrows) was surrounded by diffuse hyperintense area in T2W at week 2, but no abnormal signal was found at week 0. MRI images of femurs in T1 and T2 weight from the sham group (n = 4) were obtained at week 0 and week 2 after vehicle injection, and displayed no abnormal SI. (B) Histological analyses of distal femurs from each group (n = 4). H&E staining after steroid-induced osteonecrosis showed necrotic mass, enlarged fat cells and massive empty osteocyte lacunae (black arrows) in the SAON group. The ER on T2 weight MR images (C) and the area of abnormal SI (D) were measured for the quantification of MR images. The percentage of empty osteocyte lacunae (E) and the mean fat cell diameter (F) were measured. Data are presented as mean ± SD, and error bars in the figure denote SD, **p<0.01,*p<0.05.</p

Representative 3D micro-CT images and biomechanical compression test showed that PTH enhanced osteogenesis in the tunnel of CD.

<p>(A) Representative 3D micro-CT images reveal new bone formation within the tunnel of each group at 6 weeks after CD. (B-G) Quantitative micro-CT show the mean volumetric bone mineral density (vBMD), bone tissue volume density, connective density, trabecular number, trabecular separation, and structure model index. (H-I) Peak load and stiffness of the bone tunnels from each group are presented. n = 8 in the SAON-CD-PTH group, n = 8 in the SAON-CD group, n = 4 in the sham-CD group. Data are presented as mean ± SD, and error bars in the figure denote SD, *p<0.05, **p<0.01.</p